With Dr. John (Jay) Schneekloth (Chemical Biology Laboratory, CCR), we are attempting to develop inhibitors/chemical probes of Ubc9, the lone SUMO-conjugating enzyme in humans. SUMOylation regulates a wide variety of biological processes, and defects in SUMO homeostasis are associated with many diseases including cancer. High-resolution crystal structures of Ubc9 with drug-like ligands are being sought and obtained by fragment screening and other complementary approaches. Human tyrosyl-DNA phosphodiesterase 1 (Tdp-1) is the focus of another project. Tdp1 inhibitors are expected to act synergistically with topoisomerase 1-targeting drugs (camptothecins, indenoisoquinolines), topoisomerase 2 inhibitors (etoposide, doxorubicin), bleomycin, and DNA alkylating agents that are already used for cancer chemotherapy. In collaboration with Dr. Yves Pommier (Developmental Therapeutics Branch, CCR), we are seeking to elucidate the structural basis for inhibition of Tdp-1 by various compounds and, by extension, gain insight into how these inhibitors might be improved by rational design. Protein phosphorylation is a reversible event that is carefully controlled by the opposing activities of kinases and phosphatases. Abnormal changes to this equilibrium often lead to deleterious effects such as deregulated cell growth and cancer. We are collaborating with Dr. Terrence Burke Jr. (Chemical Biology Laboratory, CCR), an expert in the field of protein tyrosine phosphatase (PTPase) inhibition, to develop inhibitors of PTPase epsilon, a membrane-bound enzyme that is up-regulated in human breast cancer cells and which is also a negative regulator of insulin receptor signaling. To facilitate this goal, we have recently determined high-resolution crystal structures of both the membrane-proximal catalytic domain and the membrane-distal inactive PTPase domain. Crystal structures of the dual specificity phosphatases DUSP7 and DUSP22 have also been determined. The Middle East Respiratory Syndrome Coronavirus [MERS-CoV] is a highly pathogenic virus that causes severe respiratory illness accompanied by multi-organ dysfunction, resulting in a case fatality rate of approximately 40%. Like other coronaviruses, the majority of the positive-stranded RNA MERS-CoV genome is translated into a long polyprotein that is cleaved at three sites by a papain-like protease and at 11 sites by a 3C-like protease (3CLpro). Since 3CLpro is essential for viral replication, it is a leading candidate for therapeutic intervention. To accelerate the development of 3CLpro inhibitors, we determined three crystal structures of the catalytically inactive MERS-CoV 3CLpro enzyme. Current effort is focused on identifying inhibitors of this enzyme by fragment screening. Extramural collaborators include investigators at the United States Army Medical Institute of Infectious Diseases (Drs. Robert Ulrich and Kamal Saikh), with whom we are studying the role of the adaptor molecule MyD88 in the innate immune response and structure/function relationships in dual-specificity phosphatases.